Limits...
Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

Show MeSH

Related in: MedlinePlus

Multimerization assays with the Cupidin deletion mutants. Cross-linking assay using the chemical cross-linker DMP. Bacterially expressed GST-proteins were digested by thrombin, and the resulting untagged proteins were incubated in the presence (+) or absence (-) of DMP followed by Western analysis using anti-Cupidin (CPD C) antibody. One asterisk indicates the migration of multimers, and two asterisks indicate that of monomers. (B) Co-immunoprecipitation assay using extracts from cells in which the three distinct constructs shown in the upper panels were heterologously overexpressed in COS7 cells. I, Input; C, IP samples using non-immune serum; F, IP samples using anti-Flag antibody. Western blot analysis was performed using anti-Cupidin antibody, and the multimerization efficacies were calculated from the band signal intensities, and shown in the graph at the bottom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666743&req=5

Figure 2: Multimerization assays with the Cupidin deletion mutants. Cross-linking assay using the chemical cross-linker DMP. Bacterially expressed GST-proteins were digested by thrombin, and the resulting untagged proteins were incubated in the presence (+) or absence (-) of DMP followed by Western analysis using anti-Cupidin (CPD C) antibody. One asterisk indicates the migration of multimers, and two asterisks indicate that of monomers. (B) Co-immunoprecipitation assay using extracts from cells in which the three distinct constructs shown in the upper panels were heterologously overexpressed in COS7 cells. I, Input; C, IP samples using non-immune serum; F, IP samples using anti-Flag antibody. Western blot analysis was performed using anti-Cupidin antibody, and the multimerization efficacies were calculated from the band signal intensities, and shown in the graph at the bottom.

Mentions: To assess the multimerization ability of these Cupidin deletion mutants, the GST-proteins were treated with thrombin to remove the GST moiety, which are known to bind each other, and was subjected to a mobility shift assay using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with the cross-linker dimethyl pimalidate (DMP). Upon DMP treatment, immunoreactivity for CPDΔ191–230 containing both LZA and LZB was enhanced at a position indicative of multimers in comparison with that for CPDΔ191–283 and CPDΔ231–283, both of which contained LZB only (Fig. 2A). To test a possible correlation between multimerization and Cdc-42 binding ability, we coexpressed both GFP- and Flag-tagged constructs of CPD or CPDΔ191–230 in COS cells together with either dominant-active Cdc42 (Cdc42V12) or dominant-negative Cdc42 (Cdc42N17), followed by evaluation of the interaction between GFP- and Flag-tagged constructs using immunoprecipitation with an anti-Flag antibody (Fig. 2B). With both CPD and CPDΔ191–230 constructs, GFP-tagged constructs were coimmunoprecipitated with Flag-tagged constructs in intact cells, regardless of whether they expressed active or inactive Cdc42. Although the coimmunoprecipitation efficiency of GFP-tagged CPDΔ191–230, which showed a loss of Cdc42-binding activity, was 30% lower than that of GFP-tagged CPD, these results indicated that Cdc42 binding to Cupidin is substantially independent of the self-multimerization of Cupidin.


Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Multimerization assays with the Cupidin deletion mutants. Cross-linking assay using the chemical cross-linker DMP. Bacterially expressed GST-proteins were digested by thrombin, and the resulting untagged proteins were incubated in the presence (+) or absence (-) of DMP followed by Western analysis using anti-Cupidin (CPD C) antibody. One asterisk indicates the migration of multimers, and two asterisks indicate that of monomers. (B) Co-immunoprecipitation assay using extracts from cells in which the three distinct constructs shown in the upper panels were heterologously overexpressed in COS7 cells. I, Input; C, IP samples using non-immune serum; F, IP samples using anti-Flag antibody. Western blot analysis was performed using anti-Cupidin antibody, and the multimerization efficacies were calculated from the band signal intensities, and shown in the graph at the bottom.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666743&req=5

Figure 2: Multimerization assays with the Cupidin deletion mutants. Cross-linking assay using the chemical cross-linker DMP. Bacterially expressed GST-proteins were digested by thrombin, and the resulting untagged proteins were incubated in the presence (+) or absence (-) of DMP followed by Western analysis using anti-Cupidin (CPD C) antibody. One asterisk indicates the migration of multimers, and two asterisks indicate that of monomers. (B) Co-immunoprecipitation assay using extracts from cells in which the three distinct constructs shown in the upper panels were heterologously overexpressed in COS7 cells. I, Input; C, IP samples using non-immune serum; F, IP samples using anti-Flag antibody. Western blot analysis was performed using anti-Cupidin antibody, and the multimerization efficacies were calculated from the band signal intensities, and shown in the graph at the bottom.
Mentions: To assess the multimerization ability of these Cupidin deletion mutants, the GST-proteins were treated with thrombin to remove the GST moiety, which are known to bind each other, and was subjected to a mobility shift assay using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with the cross-linker dimethyl pimalidate (DMP). Upon DMP treatment, immunoreactivity for CPDΔ191–230 containing both LZA and LZB was enhanced at a position indicative of multimers in comparison with that for CPDΔ191–283 and CPDΔ231–283, both of which contained LZB only (Fig. 2A). To test a possible correlation between multimerization and Cdc-42 binding ability, we coexpressed both GFP- and Flag-tagged constructs of CPD or CPDΔ191–230 in COS cells together with either dominant-active Cdc42 (Cdc42V12) or dominant-negative Cdc42 (Cdc42N17), followed by evaluation of the interaction between GFP- and Flag-tagged constructs using immunoprecipitation with an anti-Flag antibody (Fig. 2B). With both CPD and CPDΔ191–230 constructs, GFP-tagged constructs were coimmunoprecipitated with Flag-tagged constructs in intact cells, regardless of whether they expressed active or inactive Cdc42. Although the coimmunoprecipitation efficiency of GFP-tagged CPDΔ191–230, which showed a loss of Cdc42-binding activity, was 30% lower than that of GFP-tagged CPD, these results indicated that Cdc42 binding to Cupidin is substantially independent of the self-multimerization of Cupidin.

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

Show MeSH
Related in: MedlinePlus